A calcium-binding photoprotein is one of the proteins responsible for bioluminescence. This photoprotein emits light upon specific interaction with Ca2+. The calcium-binding photoprotein is a complex of a protein having the catalytic function of oxygenation and a peroxide of luciferin as a light emitting substrate. In the calcium-binding photoprotein, the protein having the catalytic function of oxygenation refers to as an apoprotein. The peroxide of a luciferin is coelenterazine peroxide (2-hydroperoxycoelenterazine). The calcium-binding photoprotein including aequorin, clytin-I, clytin-II, mitrocomin, obelin, etc. are known and present in coelenterates. Among these photoproteins, aequorin is a photoprotein isolated from the luminous jellyfish Aequorea aequorea (1: Shimomura, In: Bioluminescence, Chemical Principles and Methods, (2006) pp 90-158, World Scientific Pub. Co.; 2: Shimomura et al., (1962) J. Cell. Comp. Physiol. 59, pp 223-240). Aequorin is a non-covalent complex of apoaequorin (21.4 kDa), which is an apoprotein, and a hydroperoxide of coelenterazine (3: Head et al., (2000) Nature, 405 372-376). Apoaequorin is composed of 189 amino acid residues in a single polypeptide chain and has three EF-hand motifs characteristic of Ca2+-binding sites (4: Inouye et al., (1985) Proc. Natl. Acad. Sci. USA. 82, 3154-3158). In the presence of Ca2+, aequorin emits blue light (λmax=˜460 nm) by an intramolecular reaction and decomposes itself into apoaequorin, coelenteramide and CO2 (5: Shimomura & Johnson (1972) Biochemistry 11, 1602-1608; 6: Shimomura & Johnson (1973) Tetrahedron Lett. 2963-2966). The complex of Ca2+-binding apoaequorin with coelenteramide obtained by this decomposition is known as blue fluorescent protein (BFP) (7: Shimomura & Johnson (1975) Nature 256, 236-238). The fluorescence and luminescence spectra of this BFP are identical to the bioluminescence spectra of aequorin (8: Shimomura (1995) Biochem. J. 306, 537-543; 9: Inouye (2004) FEBS Lett. 577, 105-110).
Recombinant aequorin can be obtained by incubating recombinant apoaequorin prepared from Escherichia coli with coelenterazine in the presence of EDTA and a reducing reagent (10: Inouye et al. (1986) Biochemistry 25, 8425-8429; 11: Inouye et al. (1989). J. Biochem. 105, 473-477). This recombinant aequorin is highly purified (12: Shimomura & Inouye (1999) Protein Express. Purif. 16, 91-95). The luminescence properties of recombinant aequorin are identical to that of native aequorin (13: Shimomura et al. (1990) Biochem. J. 270, 309-312).
Approximately 50 types of coelenterazine analogues (CTZ analogues) were hitherto synthesized and some of them were actually used to prepare semi-synthetic aequorins (e.g., 13: Shimomura et al. (1990) Biochem. J. 270, 309-312, 14: Shimomura O. et al. (1988) Biochem. J. 251, 405-410, 15: Shimomura O. et al. (1989) Biochem. J. 261, 913-920, 16: Inouye S. & Shimomura O. (1997) Biochem. Biophys. Res. Commun. 233, 349-353).
The crystal structures of aequorin and semi-synthetic aequorins have been determined (3: Head et al. (2000) Nature 405, 372-376; 17: Toma et al. (2005) Protein Science 14, 409-416), and the binding properties of Mg2+ to EF-hand motif of aequorin were also investigated by NMR analysis (18: Ohashi et al. (2005) 1 J. Biochem. 38, 613-620).
Recently, BFP was quantitatively prepared from the purified recombinant aequorin (9: Inouye, FEBS Lett. 577 (2004) 105-110; 19: Inouye & Sasaki, FEBS Lett 580 (2006) 1977-1982). BFP was found to have a substantial luminescence activity, catalyzing the oxidation of coelenterazine like a luciferase. The luminescence activity of BFP is about 10 times higher than that of Ca2+-binding apoaequorin (9: Inouye, FEBS Lett. 577 (2004) 105-110). Thus, BFP is a novel bifunctional protein having both fluorescence and luciferase activities. BFP is further converted into green fluorescent protein (gFP) having the fluorescence emission maximum peak at around 470 nm by the treatment of EDTA.
gFP is a non-covalent complex of apoaequorin with coelenteramide and aequorin can be obtained by incubation of gFP with coelenterazine at 25° C. in the absence of a reducing reagent (9: Inouye (2004) FEBS Lett. 577, 105-110). By incubation of BFP or gFP with various coelenterazine analogues in the presence of EDTA and dithiothreitol (DTT), semi-synthetic aequorins could be also prepared (19: Inouye & Sasaki (2006) FEBS Lett. 580, 1977-1982). Furthermore, the luminescence activity of BFP as a luciferase is stimulated by the addition of imidazole at the concentrations of 30 to 300 mM using coelenterazine and its analogue as a substrate (20: Inouye & Sasaki (2007) Biochem. Bioplys. Res. Commun. 354, 650-655).
In the development of use of BFP and gFP, there are some problems that the catalytic domain for oxygenation of coelenterazine, which is important basic information, or amino acid residues in BFP and gFP still remain to be elucidated. In order to solve these problems and in the development of use, it is necessary to easily prepare several tens milligrams of BFP and gFP and succeed. The present inventors have also established a method for preparing semi-synthetic gFP and semi-synthetic BFP from apoaequorin and chemically synthesized coelenteramide (21: Inouye & Hosoya (2009) Biochem. Biophys. Res. Commun. 386, 617-622). Semi-synthetic BFP prepared by this method shows a luciferase activity using coelenterazine as a substrate, similar to BFP prepared from aequorin by Ca2+-triggered luminescence reaction. The emission spectrum of semi-synthetic BFP is blue with around 470 nm.
When/As an in vivo probe to be used, a probe having the maximum wavelength of fluorescence closer to that of the near infrared region (longer than 600 nm) has been desired. In semi-synthetic BFP and semi-synthetic gFP prepared from coelenteramide analogues and apoaequorin from native aequorin, however, those having a maximum wavelength of fluorescence at 485 nm or longer have not yet been reported.